Preparation And Storage
Recommended Assay Procedures
Bioimaging
1. Seed the cells in appropriate culture medium at ~10,000 cells per well in a BD Falcon™ 96-well Imaging Plate (Cat. No. 353219) and culture overnight.
2. Remove the culture medium from the wells, and fix the cells by adding 100 μl of BD Cytofix™ Fixation Buffer (Cat. No. 554655) to each well. Incubate for 10 minutes at room temperature (RT).
3. Remove the fixative from the wells, and permeabilize the cells using either BD Perm Buffer III, 90% methanol, or Triton™ X-100:
a. Add 100 μl of -20°C 90% methanol or Perm Buffer III (Cat. No. 558050) to each well and incubate for 5 minutes at RT.
OR
b. Add 100 μl of 0.1% Triton™ X-100 to each well and incubate for 5 minutes at RT.
4. Remove the permeabilization buffer, and wash the wells twice with 100 μl of 1× PBS.
5. Remove the PBS, and block the cells by adding 100 μl of BD Pharmingen™ Stain Buffer (FBS) (Cat. No. 554656) to each well. Incubate for 30 minutes at RT.
6. Remove the blocking buffer and add 50 μl of the optimally titrated primary antibody (diluted in Stain Buffer) to each well, and incubate for 1 hour at RT.
7. Remove the primary antibody, and wash the wells three times with 100 μl of 1× PBS.
8. Remove the PBS, and add the second step reagent at its optimally titrated concentration in 50 μl to each well, and incubate in the dark for 1 hour at RT.
9. Remove the second step reagent, and wash the wells three times with 100 μl of 1× PBS.
10. Remove the PBS, and counter-stain the nuclei by adding 200 μl per well of 2 μg/ml Hoechst 33342 (e.g., Sigma-Aldrich Cat. No. B2261) in 1× PBS to each well at least 15 minutes before imaging.
11. View and analyze the cells on an appropriate imaging instrument.
Bioimaging: For more detailed information please refer to http://www.bdbiosciences.com/support/resources/protocols/ceritifed_reagents.jsp
Western blot: For more detailed information please refer to http://www.bdbiosciences.com/pharmingen/protocols/Western_Blotting.shtml
Product Notices
- Since applications vary, each investigator should titrate the reagent to obtain optimal results.
- Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
- This antibody has been developed and certified for the bioimaging application. However, a routine bioimaging test is not performed on every lot. Researchers are encouraged to titrate the reagent for optimal performance.
- Caution: Sodium azide yields highly toxic hydrazoic acid under acidic conditions. Dilute azide compounds in running water before discarding to avoid accumulation of potentially explosive deposits in plumbing.
- Source of all serum proteins is from USDA inspected abattoirs located in the United States.
- Triton is a trademark of the Dow Chemical Company.
Companion Products
Paxillin, a focal adhesion protein, is a substrate for several tyrosine kinases such as src, FAK, and p120BRC/ABL. The tyrosine phosphorylation of paxillin is affected by conditions that change cell-cell adhesion. This is consisent with the possibility that paxillin is involved in the regulation of cell morphology. Additionally, because of its SH3-binding domain, paxillin associates tightly with FAK and Crk in an extracellular matrix-independent manner. Although paxillin was initially detected in fibroblasts, its phosphorylation may be important during neurite extension during differentiation.
Development References (5)
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Ku H, Meier KE. Phosphorylation of paxillin via the ERK mitogen-activated protein kinase cascade in EL4 thymoma cells. J Biol Chem. 2000; 275(15):11333-11340. (Biology: Immunoprecipitation, Western blot). View Reference
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Leventhal PS, Feldman EL. Tyrosine phosphorylation and enhanced expression of paxillin during neuronal differentiation in vitro. J Biol Chem. 1996; 271(11):5957-5960. (Biology). View Reference
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Salgia R, Li JL, Lo SH, et al. Molecular cloning of human paxillin, a focal adhesion protein phosphorylated by P210BCR/ABL. J Biol Chem. 1995; 270(10):5039-5047. (Biology). View Reference
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Takayama Y, Tanaka S, Nagai K, Okada M. Adenovirus-mediated overexpression of C-terminal Src kinase (Csk) in type I astrocytes interferes with cell spreading and attachment to fibronectin. Correlation with tyrosine phosphorylations of paxillin and FAK. J Biol Chem. 1999; 274(4):2291-2297. (Biology: Immunofluorescence, Immunoprecipitation, Western blot). View Reference
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Turner CE, Glenney JR Jr, Burridge K. Paxillin: a new vinculin-binding protein present in focal adhesions. J Cell Biol. 1990; 111(3):1059-1068. (Biology). View Reference
Please refer to Support Documents for Quality Certificates
Global - Refer to manufacturer's instructions for use and related User Manuals and Technical data sheets before using this products as described
Comparisons, where applicable, are made against older BD Technology, manual methods or are general performance claims. Comparisons are not made against non-BD technologies, unless otherwise noted.
For Research Use Only. Not for use in diagnostic or therapeutic procedures.